Biochemistry 2006, 45, 14933-14943
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Substrate Recognition by the Hetero-Octameric ATP Phosphoribosyltransferase from Lactococcus lactis† Karen S. Champagne,‡,§ Elise Piscitelli,| and Christopher S. Francklyn*,‡,| Department of Microbiology and Molecular Genetics and Department of Biochemistry, Health Sciences Complex, UniVersity of Vermont, B403 GiVen Building, 89 Beaumont AVenue, Burlington, Vermont 05405 ReceiVed August 30, 2006; ReVised Manuscript ReceiVed October 13, 2006
ABSTRACT: Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms and a hetero-octameric form largely restricted to bacteria. Heterooctameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form and characterized the active site by mutagenesis. The KmPRPP (18.4 ( 3.5 µM) and kcat (2.7 ( 0.3 s-1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 ( 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be noncompetitive (Ki ) 81.1 µM) and competitive (Ki ) 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments aimed at investigating the side chains recognizing PRPP showed that 5′-phosphate contacts (T159A and T162A) had the largest (25- and 155-fold, respectively) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety or contacts to the 2′-OH group. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters and employ mechanistic strategies reminiscent of the broader PRT superfamily.
ATP phosphoribosyltransferase (ATP-PRT,1 EC 2.4.2.17) catalyzes the first and highly regulated step of histidine biosynthesis, which involves nucleophillic attack of N1 of ATP on C1′ of 5-phosphoribosyl-1 pyrophosphate (PRPP) to form N-1-(5′-phosphoribosyl)-ATP (PR-ATP, Figure 1A) (1). The resulting product is converted through an additional nine reactions into histidine by a pathway that is regulated both by its end product and by AMP and ADP (2). The tight regulation of this pathway (reviewed in ref 3) reflects the high energetic costs associated with histidine synthesis and the need to dramatically slow pathway flux when histidine is present in the growth media (4). Superimposed on top of † This work was supported by NIH Grant GM54899 (C.S.F.) and the Department of Energy Experimental Program to Stimulate Competitive Research (DOE-EPSCOR). * To whom correspondence should be addressed: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Ave., Burlington, VT 05405. Phone: (802) 656-8450. Fax: (802) 862-8229. E-mail:
[email protected]. ‡ Department of Microbiology and Molecular Genetics. § Current address: Department of Molecular, Cell, and Developmental Biology, 406 Sinsheimer Labs, University of California, Santa Cruz, CA 95064. | Department of Biochemistry. 1 Abbreviations: AICAR, 5′-phosphoribosyl-4-carboxyamide-5-aminoimidazole; ATP-PRT, ATP phosphoribosyltransferase; PRT, phosphoribosyltransferase; HisRS, histidyl-tRNA synthetase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; OPRT, orotidine phosphoribosyltransferase; QPRT, quinolate phosphoribosyltransferase; PRPP, 5-phosphoribosyl-1-pyrophosphate; PR-ATP, N-1-(5′-phosphoribosyl)ATP.
metabolic control of histidine biosynthesis is genetic regulation of the expression of histidine biosynthetic enzymes, by use of the classic attenuation mechanism (reviewed in ref 5). ATP-PRT is a member of the phosphoribosyltransferase (PRT) superfamily of enzymes, all of which share a common chemistry that involves transfer of the phosphoribosyl group to a nucleotide base or, in the case of glutamine phosphoribosyl pyrophosphate amidotransferase, to free ammonia generated on the enzyme (6). PRTs are found in many essential pathways, including biosynthesis and salvage of nucleotides and the synthesis of cofactors and amino acids (7). There are at least four different structurally unrelated subfamilies of PRTs. Type I PRTs, which include the adenine, orotate (OPRT), and hypoxanthine/guanine PRTs (HGPRT), are composed of four different domains, including a core region that binds both the PRPP and the nucleotide substrate, a hood domain that recognizes the nucleotide substrate, a flexible loop that closes over PRPP, and a C-terminal arm that provides dimerization for some PRTs (8). Considerable research attention has focused on the type I PRTs, as the inherited Lesch/Nyan syndrome and orotic aciduria involve respective defects in the HPRT and orotate PRTs (7). Other type I PRTs represent targets for therapeutics directed against malaria, Chagas disease, and other pathogenic eukaryotes. Notably, type I PRTs share the PRPP binding motif, a 13-residue signature sequence that is important for substrate recognition (9). The type II and type
10.1021/bi061802v CCC: $33.50 © 2006 American Chemical Society Published on Web 11/22/2006
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Champagne et al. structures of the hetero-octameric ATP-PRT have been reported, a histidine-inhibited complex of the enzyme from Thermatoga maritima (18) and ATP-activated and PRPPbound forms of the enzyme from Lactococcus lactis (19). The sequence conservation between HisGS and HisGL indicates a conserved active site and suggests a similarly conserved reaction mechanism (Figure 2). Early work characterizing the steady state kinetics of the long form indicated that the reaction proceeds by an ordered bi-bi mechanism with ATP leading (20). Another study involving determination of kinetic isotope effects of the HisGL enzyme provided evidence that the ATP-PRT transition state has ribooxocarbenium character (21). In contrast, the kinetic properties of the hetero-octameric form have not yet been characterized. As part of efforts to compare the structure and function of the hexameric and hetero-octameric versions of ATP-PRT, we report here the steady state kinetics of the hetero-octameric HisG/HisZ enzyme from L. lactis and a mutational analysis of the contacts between PRPP and the ATP-PRT based on the structure of the activated form of the complex (19). Analysis of the resulting mutant proteins highlights the importance of active site contacts with PRPP on ground state binding, as well as the interdependence of the recognition of PRPP, ATP, and the inhibitor histidine. EXPERIMENTAL PROCEDURES
FIGURE 1: ATP phosphoribosyltransferase reaction catalyzed by the hetero-octameric ATP-PRT. (A) Reaction scheme illustrating the nucleophilic attack of N1 from ATP on C1′ of PRPP. (B) Ribbon diagram of the PRPP-bound complex of the heterooctameric HisZ-HisG PRT from L. lactis (19). The four HisGS subunits are colored green, whereas the HisZ subunits are colored cyan. PRPP and inorganic phosphate are rendered in ball-and-stick mode in red and yellow, respectively.
III enzymes are represented by quinolinate (QPRT) and anthranilate PRT, respectively, which participate in NAD+ and tryptophan biosynthesis (10, 11). ATP-PRTs represent a fourth class of PRTs and comprise two different subfamilies with distinctly different quaternary structures. The hexameric ATP PRTs, termed HisGL or long form, are found in enteric bacteria and lower eukaryotes (12). Each HisGL subunit is composed of three domains (13, 14). The first two constitute a mixed R/β bilobal catalytic domain that resembles proteins of the periplasmic binding protein family (15), while the third domain provides a binding site to allow feedback inhibition by histidine (13). The two different HisGL structures that are available include a histidine/AMP-inhibited form from Mycobacterium tuberculosis (13) and an enzyme-product complex of PR-ATP and the enzyme from Escherichia coli (14). Enzymes from the second ATP-PRT family are hetero-octamers, composed of four HisGS subunits and four HisZ regulatory subunits (Figure 1B) (16, 17). The latter are structurally related to the catalytic domain of histidyl-tRNA synthetase and provide a regulatory domain to compensate for the absence of the C-terminal domain found in HisGL. Notably, both subunits are required to form a functional complex (17). Two
Construction and Purification of Mutant Proteins. Mutant versions of the L. lactis HisZ/HisG ATP-PRT were constructed in the pQE30 expression construct background (17). The T159A, T162A, S140A, K8A, K50A, and D155A mutations were introduced by use of the QuikChange mutagenesis procedure (Stratagene). In all cases, the doublestranded primers were 45 nucleotides in length. After mutagenesis, the presence of the directed mutations and the absence of unprogrammed mutations were confirmed by sequencing of the entire genes. The wild-type and all mutant proteins were expressed and purified from E. coli overexpression strains by use of a previously published protocol featuring the three-column sequence of Ni-NTA, Superdex 200, and hydroxyapatite chromatography (16). Pooled fractions from the final hydroxyapatite column were concentrated to ∼6 mg/mL, dialyzed into storage buffer [50 mM Na2PO4 (pH 7.5), 300 mM KCl, 10% (v/v) glycerol, and 10 mM β-mercaptoethanol], and then stored at 4 °C for kinetic experiments. Protein concentrations were determined from the extinction coefficient (102 400 M-1 cm-1) calculated from the sequences of HisZ and HisG, incorporating a weight average of the two subunit types, as described in ref 16. ATP-PRT Assay. The phosphoribosyltransferase activity of ATP-PRT was monitored at A290 as an increase in the level of formation of PR-ATP over time (22). The measurements were performed at 22 °C in 100 mM Tris-HCl (pH 8.5) containing 10 mM MgCl2, 150 mM KCl, 5 mM β-mercaptoethanol, and 2 units/mL inorganic pyrophosphatase. Purified proteins were added to the indicated concentration, and the reactions were initiated by the addition of PRPP. The absorbance was detected every 9 s for 10 min after initiation of the reaction. The baseline absorbance was established by zeroing the spectrophotometer before adding the PRPP. The extinction coefficient for PR-ATP is 3600 M-1 cm-1 (23), and was used to convert the absorbance units
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FIGURE 2: Multiple-sequence alignment of representative HisGS and HisGL sequences, overlaid with the secondary structure assignments from L. lactis HisGS. Sequences corresponding to HisGS are denoted with red braces, and sequences corresponding to the HisGL form are denoted with green braces. The alignment omits the histidine binding domain of HisGL, which is absent from HisGS. Blue circles beneath the alignment indicate residues in contact with the pyrophosphate moiety of PRPP, consisting of K8, K50, and S140. The green circle denotes D155. The T159 and T162 side chains are denoted with red circles. The following organisms are included in the alignment: Lactococcus, L. lactis; Streptococcus, Streptococcus mutans; Thermoanerobacter, Thermoanerobacter tengcongenis; Pseudomonas, Pseudomonas aeruginosa; Leptospira interrogans serovar lai str; Deinococcus, Deinococcus radiodurans; Pyrococcus, Pyrococcus furiosus; Escherichia, E. coli; Shigella, Shigella flexneri; Sulfolobus, Sulfolobus solfataricus; Saccharomyces, Saccharomyces cereVisiae; Candida, Candida albicans; Methanopyrus, Methanopyrus kandleri; Methanococcus, Methanococcus jannaschii; Brucella, Brucella melitensis; Agrobacterium, Agrobacterium tumefaciens; Arabidopsis, A. thaliana (there are two isozymes of A. thaliana).
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Champagne et al.
per minute to picomoles of product formed per second. Steady state parameters KmPRPP and KmATP were determined by varying the ATP or PRPP concentration while holding the other substrate at a fixed concentration. The PRPP kinetics were measured using a range of 10 µM to 1 mM in PRPP, with 5 mM ATP. For some mutants, the highest concentration of PRPP had to be adjusted upward to accommodate poorer binding. Similarly, the variable ATP assays were performed using ATP concentrations in the range of 100 µM to 5 mM, maintaining the PRPP concentration at 2 mM. The initial rates were calculated from the linear portions of the progress curves, and then the steady state parameters were obtained from saturation plots of V/[Eo] versus [S]. Each substrate concentration was performed three to five times such that the plots were derived using the mean value of V/[Eo]. Turnover numbers (kcat) were calculated from the quotient of the maximal catalytic rate divided by the total concentration of active sites. The kinetic parameters for all mutant proteins (except S140A, which proved to be unstable) were measured as described above. The concentrations of the mutant enzymes were adjusted upward when necessary to achieve an initial rate of PR-ATP formation that was approximately equivalent to that of the wild-type enzyme. Reaction mixtures that included inhibitors were prepared by adding AMP or histidine to the 2× reaction buffer and preincubated for 5 min in the presence of enzyme, and then the reactions were initiated by the addition of PRPP. The AMP concentrations employed in the inhibitor assays spanned a range from 0 to 10 mM, while histidine concentrations ranged from 0 to 200 µM. Data Fitting and Analysis. The kinetic parameters, including Km and Vmax, were extracted by directly fitting the initial rate versus substrate data to the Michaelis-Menten equation:
V)
Vmax[S] Km + [S]
(1)
This equation was used for experiments involving fixed PRPP and variable ATP concentrations, as well as fixed ATP and variable PRPP concentrations. The data from the histidine and AMP inhibition experiments were plotted as 1/V versus 1/[S] for each of the four different concentrations of histidine and AMP. The slopes of these lines were used to determine the Ki for histidine and AMP by plotting the Kmapp/Vmax values against histidine or AMP concentration. The slope of the resulting line is equivalent to Km/Ki, and the abscissa intercept equals the -Ki value for competitive inhibition systems:
Kmapp )
Km [I] + Km Ki
(2)
RESULTS Steady State Kinetic Parameters for the PRT Reaction Catalyzed by the Hetero-Octameric ATP-PRTase Reaction. The reaction catalyzed by ATP-PRT and a ribbon diagram of the structure of the L. lactis ATP-PRT hetero-octameric complex are presented in Figure 1. As described in Experimental Procedures, the steady state kinetic parameters were determined by use of a continuous assay in which the production of PR-ATP is monitored by the increase in
FIGURE 3: Steady state kinetics for wild-type heterooctameric ATPPRT at 22 °C and pH 8.5. (A) Plot of velocity vs varying concentrations of PRPP, determined at a constant ATP concentration of 5 mM. Wild-type ATP-PRT was added to a final concentration of 24.9 nM. (B) Plot of velocity vs varying ATP concentrations, determined at a constant PRPP concentration of 2 mM. Wild-type ATP-PRT was added to a final concentration of 27.3 nM. The experiments were performed as described in Experimental Procedures. The error bars represent the standard error of the mean for the velocity measurements, which were measured three to five times. The 10-15% errors in the individual measurements were typical for the kinetics of the wild type and all of the mutants reported here.
absorbance at 290 nm. Under conditions where the concentration of ATP was fixed at 5 mM and that of PRPP was varied, straightforward Michaelis-Menten behavior was observed, allowing kinetic parameters (kcat ) 2.67 ( 0.27 s-1 and KmPRPP ) 18.4 ( 3.5 µM) to be readily determined (Figure 3A). The Km for PRPP for the “short form” enzyme compares reasonably with the values of KmPRPP determined for “long form” ATP-PRTs (summarized in Table 1), which range from values of 11-67 µM determined for the Salmonella typhimurium enzyme (1, 20) to 130 µM determined for the enzyme from Arabidopsis thaliana (24). This value for KmPRPP is also comparable to the corresponding
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Table 1: Kinetic Parameters for Hexameric and Hetero-Octameric ATP Phosphoribosyltransferases from Various Species organism
kcat (s-1)
Km(PRPP) (µM)
Km(ATP) (mM)
Ki(His) (µM)
Ki(AMP) (mM)
ref
L. lactis T. maritima S. typhimurium S. typhimurium A. thalianab
2.67 ( 0.27 NDa NDa 2.7 NDa
18.4 ( 3.5 NDa 67 11 130/570
2.7 ( 0.26 NDa 0.2 0.11 0.60/0.51
81.1 350 100 70 45/320c
1.44 NDa 5 5 NDa
this work 18 1 20, 25 24
a Not determined. b Parameter values for the two A. thaliana isozymes (ATP-PRT1 and ATP-PRT2) are indicated on either side of the forward slash. c Reported as an IC50.
parameters for other PRTs, including the orotate PRT (Km ) 18.7 µM), quinolate PRT (KmPRPP ) 30 µM), and the HPRTs from Tritrichomonas foetus (Km ) 46 µM) and Trypanosoma cruzi (32.4 µM). Thus, similar Michaelis constants for the PRPP substrate are observed across the entire PRTs superfamily. The level of catalytic activity of the short form ATP-PRT as determined in our assay is comparable to that measured for the long form enzyme from Salmonella measured previously (20, 25) but significantly lower than those of the HPRTs from the type I PRT families, which exhibited kcat values in the range of 23-76 s-1. These enzymes lack the complex regulation of ATP-PRT and are invariably dimers. Kinetic parameters were also determined under conditions where PRPP was the constant substrate at 2 mM and ATP was the variable substrate. Over a concentration range of 100 µM to 5 mM in ATP, reliable fits to the Michaelis-Menten equation were obtained, providing the following kinetic parameters: kcat ) 2.3 ( 0.25 s-1, and KmATP ) 2.4 ( 0.24 mM (Figure 3B). Notably, this value is significantly higher than the values of KMATP reported for other ATP-PRTs. For example, the KmATP for the S. typhimurium enzyme was reported as 110-200 µM (1, 20), while values of 600 and 510 µM were determined for the two isozymes of A. thaliana (24). Nonphysiological concentrations of ATP in excess of 15 mM were inhibitory to ATPPRT, which may reflect a competition by ATP for the PRPP binding site, as proposed for AMP (14). This phenomenon remains to be investigated in more depth. Kinetic Parameters of Inhibition by Histidine and AMP. The inhibition of the L. lactis enzyme by histidine was determined from velocity versus PRPP concentration plots at a constant ATP concentration of 5 mM, and over histidine concentrations ranging from 0 to 200 µM (Figure 4A). A replot of the slopes of the reciprocal plots against histidine concentration returned a value of 81 µM, indicating that, to a first approximation, histidine is a noncompetitive inhibitor (Figure 4B). This value is in good agreement with early published values (70-100 µM) for the S. typhimurium enzyme, but there was an early discrepancy in the literature about whether this inhibition is noncompetitive or uncompetitive (1, 26). Vega et al. (18) reported that histidine is a noncompetitive inhibitor of the hetero-octameric T. maritima enzyme with a Ki of 350 ( 20 µM, while Ohta et al. reported IC50’s for inhibition by histidine of the A. thaliana ATPPRT1 and ATP-PRT2 of 45 and 320 µM, respectively (24). Noncompetitive inhibition by histidine with respect to PRPP for the short form of HisG is consistent with prior data suggesting that the histidine binding site is located in the HisZ subunit, at least 43 Å from C1′ on PRPP where chemistry occurs (19). It should be noted that reciprocal plots in Figure 4A appear to intersect below the horizontal axis, which at least formally raises the possibility that the ESI
complex has at least a fraction of the activity of the ES complex (27). A similar experimental strategy was used to determine the Ki for AMP with PRPP. As determined from the replot in Figure 4D, the Ki for AMP is 1.81 mM and represents competitive inhibition with respect to PRPP. This value is within a factor of 3 of the reported Ki for AMP (5 mM) for the enzyme from S. typhimurium (1). Mutations in the PRPP Binding Site Result in a Loss of Catalytic ActiVity. The similarities in steady state kinetic parameters between the hetero-octameric and hexameric ATP-PRTs, as well as their relationship to the parameters for the more distantly related type I PRTs, motivated a more detailed analysis of the contacts with PRPP suggested by the L. lactis crystal structure (Figure 5). The three moieties of PRPP that are directly recognized by the enzyme include the 5′-phosphate, the 2′- and 3′-ribose hydroxyl groups, and the pyrophosphate group (19). In both the long and short form enzymes, the 5′-phosphate is recognized by side chains β9 and R7 in HisG, including the main chain amides at T159 and G160, and the γOH groups of T159 and T162. The two latter residues are highly conserved in ATP-PRTs, the only exceptions being the substitution of T159 with serine in the hexameric enzymes from eukaryotes (Figure 2). Pyrophosphate recognition appears to be provided by basic and polar side chains contributed from diverse structural elements, including the β1-R1 loop, the β3-R2 loop, and the N-terminus of R6 (Figure 2). To explore the contributions of these contacts to catalysis in detail, six of the most conserved contacts, including K8, K50, S140, D155, D159, and T162, were substituted with alanine. The resulting mutant proteins were then characterized with respect to their steady state parameters. As indicated in Figure 6 and Table 2, the substitutions that proved to be the most deleterious were those associated with interactions with the 5′-phosphate. The T159A and T162A mutants were both significantly compromised for PRPP binding, with 279- and 49-fold increases in KmPRPP (Figure 6A,B). In contrast, both mutants were much less affected at the level of kcat (decreased only 2-3-fold relative to that of the wild type) and exhibited slightly decreased values for KmATP (Figure 6C,D). These latter parameters indicate that the effect on PRPP binding is likely to be a consequence of the loss of a specific contact and not the result of a global structural defect. The D155A mutant substitutes a carboxylate contact to the 2′-OH group of PRPP that is nearly invariant in both the HisGL and HisGS ATPPRT subfamilies. Of all the mutants tested in the study, D155A was the mutant least affected with respect to enzymatic activity, exhibiting no effect on kcat, and only 3.8and 1.7-fold increases in the Michaelis constants for PRPP and ATP, respectively (Table 2). Thus, this contact appears to provide an only modest (2-3 orders of magnitude (43). While ATP-PRTs and type I PRTs are defined by structurally unrelated catalytic domains, functionally homologous active site loops and secondary structure elements can be identified that appear to underlie similarities in mechanism. Thus, the PRPP loop of type I PRTs, with its characteristic pair of acidic residues and conserved TXXT motif, can be readily superimposed (rmsd ) 0.5-0.8 Å) on the PRPP loop of ATP PRTs (14). Recognition of the pyrophosphate group by type I PRTs is mediated by two elements, the “PPi loop” found between β1 and R2 that exhibits minimal sequence conservation (save for a rare nonproline cis peptide between the first and second residues of the loop) and the “flexible loop”, a conformationally mobile structural element that is only ordered when the Mg2+-PRPP moiety is bound in the active site. Notably, mutagenesis studies on type I PRTs focusing on the PPi and flexible loops have been reported and indicate that, like our results with K8A and K50A, substitutions of PRPP-contacting residues preferentially increase KmPRPP without imposing significant effects on kcat (39, 44). In a study that correlated mutations in human HPRT with various in vitro and in vivo phenotypes, among the recurring mutations were substitutions that affected residues responsible for binding Mg2+, which is coordinated to the pyrophosphate (45). One perplexing issue not yet addressed in any of the ATP-PRT structure studies or the work reported here is the absence of information concerning Mg2+ binding. Divalent metal is required for activity, but there is no structural evidence for it in any of the complexes reported thus far. As yet, none of the available ATP-PRT crystal structures has provided information about the pattern of Mg2+ binding in the type IV enzyme, so similarities in metal binding cannot be assessed. This important question remains to be addressed in future studies. ACKNOWLEDGMENT We thank Ethan Guth and Robert Hondal for useful discussions and comments on the manuscript and Tim Hunter
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